Wnt signaling plays a critical role in cell fate determination as well as tissue development and is highly conserved from C. Elegans to humans. Wnts were initially identified as a consequence of their transcriptional activation by mammary tumor virus promoter insertion, which initiates tumor formation. Later studies established that constitutively activated Wnt signaling through alterations afflicting the intracellular mediators, APC and b-catenin, is crucially involved in several types of human cancer. We have identified human breast and ovarian carcinoma cell lines with Wnt pathway activation in the absence of mutations in APC/b-catenin. Extracellular Wnt antagonists, FRP1 and DKK1 which are known to block ligand/receptor interactions, caused a dramatic downregulation of Wnt signaling, establishing the presence of an autocrine mechanism in these tumor cells. We plan to continue our investigations aimed at the characterization of this newly identified mechanism for constitutive Wnt pathway activation. Aim 1 of this proposal is directed towards determining how autocrine Wnt signaling influences the transformed phenotype of these human breast/ovarian tumor cells including their response to therapeutic agents and their stem/progenitor-like properties. Aim 2 focuses on the identification of the molecular mechanisms responsible for the establishment of Wnt autocrine signaling in these tumor cells. Aim 3 is directed toward elucidation of the effects of Wnt ligand on endogenous LRP6 receptor turnover, utilizing a highly specific and sensitive Mab newly generated by us. Moreover, studies within this Aim will be directed towards the investigation of the effects of the tumor microenvironment on "in vivo" growth of breast cancer cells over-expressing endogenous LRP6.